Mass production of entomopathogenic fungi Purpureocillium lilacinum PL1 as a biopesticide for the management of Amrasca devastans (Hemiptera: Cicadellidae) in okra plantation
Egyptian Journal of Biological Pest Control volume 33, Article number: 85 (2023)
Effective management strategies are crucial in minimizing the adverse consequences associated with the leafhopper, Amrasca devastans (Dist.) (Hemiptera: Cicadellidae). Economic limitations to entomopathogenic fungi production present a substantial challenge, particularly in developing countries. This study aimed to investigate a cost-effective solid-state fermentation (SSF) for large-scale production of Purpureocillium lilacinum PL1 conidia to manage A. devastans infestations in okra cultivation.
Rice and maize were demonstrated as highly suitable substrates for producing conidia densities of over 2 × 1010 conidia g−1. Furthermore, the influence of agricultural phytosanitary agents on the growth rates of P. lilacinum PL1 was evaluated. Certain pesticides were ineffective on the expansion of P. lilacinum PL1 colonies, while fungicides exhibited complete inhibition. The laboratory investigation revealed that 1 × 107 conidia ml−1 of P. lilacinum PL1 exhibited a success rate of 88.66% in decreasing the population of A. devastans nymphs in vitro. Furthermore, field investigations carried out in okra plantations demonstrated that the utilization of P. lilacinum PL1 at the concentration of 1 × 107 conidia ml−1 of resulted in a significant reduction of the pest nymph population by 72.87% subsequent to the 2 applications.
In conclusion, the cost-effective mass production of P. lilacinum PL1 conidia through SSF presents a promising solution for managing A. devastans infestations in okra farming, particularly in economically challenged regions.
Okra [Abelmoschus esculentus (L.) Moench. family Malvaceae], a vegetable crop primarily cultivated in warm regions of Africa and Asia including Viet Nam, is grown by smallholder farmers for its edible immature pods (Tong 2016). Leafhopper [Amrasca devastans (Dist.); Hemiptera: Cicadellidae] is a significant pest of okra, which has a high prevalence in regions characterized by tropical and subtropical climates (Rehman et al. 2015). The feeding behavior of the leafhopper on okra plants involves the consumption of phloem sap, resulting in the manifestation of symptoms such as leaf yellowing, curling, and stunting, leading to a notable decrease in crop yield. The severity of damage varies depending on the infestation level and okra variety susceptibility (Sarwar 2020). Insecticides, particularly chemical synthetic pesticides, are frequently used by farmers to control this pest. The application of pesticides has resulted in ecological contamination, the emergence of pest resistance, and potential dangers to human health (Pathak et al. 2022). Therefore, utilization of eco-friendly techniques, such as entomopathogenic fungi (EPFs), for the control of sap-sucking pests has become increasingly prevalent as a substitute for chemical insecticides (Skinner et al. 2014).
Purpureocillium lilacinum is a hyphomycete fungus known for its entomopathogenic properties, which has made it a popular biocontrol agent against various insect pests, including soil-dwelling and above-ground pests (Goffré and Folgarait 2015). The fungal synthesizes diverse enzymes that degrade organic compounds, such as proteases and chitinases, which play a crucial role in catalyzing and hydrolysis of the insect cuticle, thereby facilitating the successful infiltration and subsequent infection of the host organism (Ibrahim et al. 2016). Furthermore, it has been indicated that administration of P. lilacinum PL1 showed significant lethality against whitefly [Bemisia tabaci (Gennadius); Hemiptera: Aleyrodidae] nymphs, with mortality rates of 88.24% in the laboratory and 78.86% in the field, respectively (Nguyen et al. 2023). In addition, P. lilacinum is also known to control various other insects, including mosquitoes, beetles, thrips, fruit flies, whiteflies, aphids, and even nematodes, making it a valuable tool in integrated pest management strategies (Amala et al. 2013; Goffré and Folgarait 2015). Hence, the utilization of P. lilacinum as a biocontrol agent for A. devastans in okra farming holds promise as an eco-friendly and sustainable approach.
The high cost of producing EPFs for pest control poses a significant challenge, particularly in developing nations (Morán-Diez and Glare 2016). The employment of solid-state fermentation (SSF) has surfaced as a viable and uncomplicated approach for the economical generation of EPFs, particularly for the production of conidia (Rayhane et al. 2019). SSF utilizes various agro-industrial residues as substrates, such as cereal bran, sugarcane bagasse, and sawdust (Sadh et al. 2018). Various strains of EPFs, such as Metarhizium anisopliae, Beauveria bassiana, and Paecilomyces fumosoroseus, have been effectively cultivated through SSF by adjusting growth conditions, substrate composition, and inoculation rate to attain optimal spore production (Qiu et al. 2019).
This study aimed to investigate the feasibility of utilizing SSF to cultivate P. lilacinum PL1 conidia as a strategy for the leafhopper A. devastans management in okra fields. In addition, the compatibility of EPFs and chemical pesticides is important for integrated pest management (IPM) strategies. Certain chemical pesticides may reduce EPF efficacy, while others may enhance their activity (Abd-El-Khair et al. 2019). This study further examined the compatibility of P. lilacinum PL1 with synthetic fungicides and pesticides through cocultivation and investigated their impact on the growth of this fungus.
The study was designed using a flow diagram to visually depict the research methodology and its progression (Fig. 1). This study assessed the optimal propagation and solid fermentation mediums for the efficient production of P. lilacinum PL1 conidia. Besides that, the effect of phytosanitary substances on the growth of P. lilacinum PL1 was investigated. Additionally, the study examined the lethality of P. lilacinum PL1 on A. devastans nymphs under controlled laboratory conditions. Furthermore, the efficacy of P. lilacinum PL1 in the management of A. devastans nymph populations in okra fields was assessed (Fig. 1).
Propagation of A. devastans nymphs in the greenhouse
Greenhouse leafhopper propagation was conducted according to the protocol described by Manivannan et al. 2018. The leafhopper A. devastans population was obtained from okra plantations and subsequently cultivated in a greenhouse to facilitate population growth. The maintenance of the leafhopper culture was achieved through the cultivation of okra plants in pots that were enclosed with a cage made of a thin film of mylar (Manivannan et al. 2018). The confirmation of the final instar of A. devastans nymphs was conducted based on the pictorial guide (Nagrare et al. 2012).
The fungal strain P. lilacinum PL1 was isolated from B. tabaci cadavers, obtained from a previous study (Nguyen et al. 2023), and was maintained in Potatoes dextrose agar (PDA) culture medium (HiMedia, India) at 30 °C.
Effect of culture medium on primary multiplication of P. lilacinum PL1
The mycelia disk measuring 5 mm in diameter of P. lilacinum PL1 was subjected to cultivation on various nutrient media, including Potato Dextrose Agar (PDA), Malt Agar (MA), Czapek-Dox (CZ), and Sabouraud Agar (SA). The Petri dishes were subjected to incubation at a temperature of 28 °C for a duration of 7 days. The extent of radial growth of the fungus was quantified by measuring the diameter of the colony. The experiment was conducted in triplicate to ensure the accuracy of the results.
Effectiveness of fungicides and insecticides on the growth of P. lilacinum PL1
As shown in Table 1, the pesticides or fungicides were integrated into a PDA medium in this investigation. The PDA medium was sterilized at 121 °C for 20 min. Following, the recommended field doses (Table 1) of pesticides or fungicides were added and evenly distributed in Petri plates with a diameter of 90 mm, with a volume of 18 ml/plate. Plugs with a diameter of 5 mm were obtained from the periphery of the P. lilacinum PL1 colonies and then transferred to the PDA medium supplemented with pesticides or fungicides. The control group consisted of P. lilacinum PL1 cultured on free-pesticide PDA culture medium (Mayo-Prieto et al. 2022). Subsequently, the plates were incubated at 25 °C in the absence of light for a duration of 7 days. The mycelial growth of P. lilacinum PL1 was quantified following a 7-day cultivation, and the experimental procedures were conducted in triplicate. The assessment of the initial toxicity of fungicides and insecticides on the growth of P. lilacinum PL1 was conducted utilizing a 4-level category system. The categories were defined as follows: 1—harmless, indicating an inhibition less than 50%; 2—slightly harmful, indicating an inhibition ranging from 50 to 79%; 3—moderately harmful, indicating an inhibition ranging from 80 to 99%; and 4—harmful, indicating an inhibition greater than 99% (Hassan et al. 1985).
Effect of substrate on the production of P. lilacinum PL1 conidia utilizing solid-state fermentation
Mass multiplication of P. lilacinum PL1 through solid-state fermentation was conducted using 4 different solid substrates: rice, paddy seeds, maize, and bran (Sadh et al. 2018). The experimental procedure involved the uniform allocation of 1 kg of distinct substrates onto a tray with dimensions of 40 cm × 40 cm. Subsequently, 500 ml of water was added, and the tray was subjected to sterilization through autoclaving. Conidia of P. lilacinum PL1 were acquired from PDA culture medium, then suspended in phosphate-buffered saline (PBS), and subsequently introduced to the sterilized substrate at a density of 1 × 106 conidia g−1. Subsequently, the trays underwent an incubation process at 28 °C for 14 days. Following the incubation, the conidia density of P. lilacinum PL1 in each substrate was assessed through either serial dilution on PDA plates or enumeration with a hemocytometer (Hirschmann, MO, USA).
Lethality of P. lilacinum PL1 on A. devastans nymphs in vitro
A total of 50 last instar nymphs of A. devastans were gathered and transferred onto okra leaves, which were then arranged in a 150-mm-diameter Petri dish. Each dish contained 50 nymphs. The P. lilacinum PL1 conidia were obtained from rice substrate following 14 days of cultivation. These conidia were then suspended in sterile distilled water containing Tween 80 (0.02% v/v) and adjusted to a concentration of 1 × 107 conidia ml−1 (Nguyen et al. 2023). A toxicity investigation was conducted wherein A. devastans nymphs were administrated with a 5 ml of P. lilacinum PL1 conidia suspension. The group only received water supplemented with 0.02% Tween 80 was referred to as an untreated group. In contrast, the positive control group was subjected to a treatment consisting of 5 ml of Abamectin (AM) at a concentration of 250 ppm (v/v). Following the air-drying, the leaves were subsequently relocated to a separate Petri dish that was furnished with a layer of 1.5% agarose gel and incubated at 28 °C, accompanied by a relative humidity range of 50–60% (Shah et al. 2020). Daily monitoring of the nymph's mortality was conducted over a period of 7 consecutive days. The calculation of mortality was performed as a percentage utilizing Abbott's formula (Abbott 1925) as follows:
where m and n are for the percentages of dead nymphs in the treatment group and untreated group, respectively.
Evaluation of the lethality of P. lilacinum PL1 on A. devastans nymphs under field conditions
Field studies utilizing a randomized complete blocks design (RCBD) were carried out at three distinct okra farms located in Cu Chi, Ho Chi Minh City, Vietnam (Fig. 2). Each treatment was replicated 3 times. The study utilized an experimental design consisting of plots with dimensions of 10 m by 5 m and a spacing of 1 m. Each plot was capable of accommodating a total of 50 okra plants. Before the treatment, the quantification of A. devastans nymphs was carried out on okra leaves situated at the top, middle, and bottom of each plant/plot. The experimental interventions comprised P. lilacinum PL1 at a concentration of 1 × 107 conidia ml−1, Abamectin (AM) at a concentration of 250 ppm (v/v) as a positive control, and untreated plots sprayed with water as negative controls with spray volume of 300 l/hectare were utilized to apply two sprays at 7-day intervals during the afternoon after 4 pm (Lavers 2001). The enumeration of A. devastans nymphs was carried out on the leaves located at the top, middle, and bottom portions of every plant/plot following a 7-day and 14-day treatment. The Henderson–Tilton formula was employed to assess the effectiveness of each treatment in managing the A. devastans nymph population (Henderson and Tilton 1955).
where n: insect population; T: treated; Co: control.
Results were calculated using the mean of triplicate readings.
The study utilized a completely randomized design (CRD) with 3 replicates for each treatment. Data were analyzed using SAS 9.4 software (SAS, Inc., Cary, NC, USA) and presented as the mean ± standard error of the mean based on triplicate readings. Statistical significance between groups was determined using Duncan’s test, with p < 0.05 indicating significance. Probit analysis was conducted using SAS 9.4 software to calculate LT50 and LT90.
Effect of culture medium on the growth of P. lilacinum PL1 mycelia
The primary multiplication of P. lilacinum PL1 was conducted using four nutrient media including PDA, MA, CZ, and SA. The growth rate of P. lilacinum PL1 mycelia was assessed by measuring its radial expansion after 7 days of cultivation. The results obtained indicate that there were significant variations in the growth rates of P. lilacinum PL1 among the culture media that were investigated (p < 0.05). Significantly, the most substantial growth rates were observed on MA and PDA media, with mycelia diameters ranging from 69.7 to 77.0 mm. In contrast, the CZ medium exhibited a comparatively lower growth rate, as evidenced by a mycelial diameter of 45.5 mm (Fig. 3A, B).
Effectiveness of fungicides and insecticides on the growth of P. lilacinum PL1 mycelia
The effectiveness of fungicides and insecticides on the growth of P. lilacinum PL1 was evaluated over a period of 14 days. The results, as depicted in Fig. 4A, B, showed that HC, PP, and CZ had a considerable effect on fungal growth, with complete inhibition of the growth of P. lilacinum PL1 colonies. The level of inhibition was categorized as level 4, indicating the highest level of impact—harmful (Table 2). In contrast, CuNPs alone had a minimal effect on the proliferation of fungi, resulting in an inhibition rate of approximately 7.24%, which was categorized as level 1or harmless (Table 2). In case of insecticides, AM had a slight effect on the growth of P. lilacinum PL1 (Fig. 4A, B), with a 67.29% inhibition rate of the fungal colony growth, categorized as level 2 or slightly harmful (Table 2). On the other hand, CM, DF, and TM had minimal effects on fungal growth (Fig. 4A, B), with less than 50% inhibition, classified as level 1 or harmless (Table 2).
Effect of solid substrate on the mass production of P. lilacinum PL1 conidia
The effect of solid substrate on the mass propagation of P. lilacinum PL1 conidia was investigated through the utilization of 4 organic substrates, namely rice, rice husk, maize, and paddy seeds. The fermentation was carried out for 14 days, wherein the growth rate of P. lilacinum PL1 conidia was observed. The results as shown in Fig. 5 suggested that the substrate significantly affects the biomass yield of P. lilacinum PL1 conidia. Obtained results revealed that the growth of P. lilacinum PL1 was most favorable on rice and maize substrates, yielding approximately 2 × 1010 conidia g−1, following a 14-day cultivation period (Fig. 5). In contrast, a significantly low conidial mass of 2.2 × 109 conidia g−1 was seen in paddy seeds substrate (Fig. 5).
Lethality of P. lilacinum PL1 on A. devastans nymphs in vitro
The effect of P. lilacinum PL1 on A. devastans mortality was examined through an in vitro study. The data in Fig. 4A demonstrated that the application of P. lilacinum PL1 at the concentration of 1 × 107 conidia ml−1 resulted in a significant reduction in the population of A. devastans nymphs within a 7-day treatment. The treatment exhibited an efficacy rate of 86.66%, which did not exhibit a significant difference compared to the efficacy rate of the AM treatment group. Despite P. lilacinum PL1 taking longer to eliminate A. devastans nymphs than AM, with LT50 values of 4.21 and 2.14 days, respectively (Table 3), their lethality became comparable after 7 days of treatment. Microscopic examination of A. devastans nymph cadavers in the P. lilacinum PL1 treatment group revealed the presence of hyphae on the cuticle, displaying microscopic characteristics consistent with indications of P. lilacinum infection, as depicted in Fig. 6B and C (Nguyen et al. 2023).
Application of P. lilacinum PL1 reduced A. devastans nymph’s populations on okra plantations
The efficient usage of P. lilacinum PL1 in the control of A. devastans nymphs was further investigated on okra plantations in Cu Chi, Ho Chi Minh City, Vietnam (Fig. 7). The investigation was conducted within defined environmental parameters, including a temperature range of 28–36 °C, with air humidity levels were 55–70% RH, and no precipitation during the study. Before treatment, the A. devastans nymph densities on okra leaves ranged from 6.45 to 6.90 nymphs/leaf (Table 4), highlighting the necessity for prompt implementation of control strategies. The fungus P. lilacinum PL1 was administered at a concentration of 1 × 107 conidia ml−1, and AM was utilized as a positive control at a concentration of 250 ppm (v/v), in accordance with the instructions provided by the manufacturer.
The application of AM and P. lilacinum PL1 sprays resulted in a significant reduction in the population density of A. devastans nymphs than the control group. Following 7 days subsequent to the initial treatment, P. lilacinum PL1 exhibited greater efficacy in the control of A. devastans nymphs in comparison with AM, with efficacy rates of 64.46 and 32.22%, respectively (Table 4). Subsequent treatment was conducted 7 days after the initial treatment. According to the findings presented in Table 4, the density of A. devastans nymphs in the untreated group exhibited an increase to 7.96 nymphs/leaf after 7 days of the second treatment. However, in the groups that received AM and P. lilacinum PL1 treatment, the nymph density was observed to be 4.95 and 2.27 nymphs/leaf, and corresponding efficiencies were 34.67 and 72.87%, respectively (Table 4).
Biopesticides derived from microbes are becoming more popular worldwide due to concerns about agrochemicals and demand for organic food. However, to ensure sustainable agricultural production, effective mass production and field application of biopesticides are necessary (Olson 2015). The selection of growth medium significantly impacts the primary cultivation of biocontrol agents, including P. lilacinum PL1. The growth rates of P. lilacinum PL1 mycelia were significantly affected by the nutrient media utilized, according to our research. MA and PDA media exhibit superior growth rates, whereas CZ medium yields weaker growth, indicating inadequate nutrient levels or unfavorable environmental conditions for the fungus. Furthermore, suitable fermentation substrates must also be considered for conidia production, taking into account factors such as availability and cost for conidia production (Hynes et al. 2006). Previous studies indicated that low-cost substrates such as rice, corn, and wheat brans can be used to mass production of different Paecilomyces spp. conidia, while other substrates like dried banana leaf, sorghum grains, used tea leaves, wheat bran-sawdust, and wheat bran-malt sprout mixture were suitable for high-density propagule production of Trichoderma spp. (Rini and Sulochana 2008). In this study, the growth of P. lilacinum PL1 on different organic materials in Viet Nam was investigated. It was indicated that P. lilacinum PL1 grew on all 4 solid substrates, but the level of colonization and biomass production varied. Rice and maize were the most suitable substrates for P. lilacinum PL1 conidia production, with densities exceeding 2 × 1010 conidia g−1. This finding was consistent with a previous study that found P. fumosoroseus, isolated from dead lepidopteran caterpillars in India, produced more conidia when cultivated on sorghum than on corn, rice, pearl millet, or wheat (Sahayaraj and Namasivayam 2008).
Currently, agriculture utilizes an integrated approach that combines chemical compounds, cultural measures, resistant varieties, and biocontrol agents to achieve environmental sustainability. However, it is crucial to assess the potential interactions between chemical products and the development of biological agents. The present study investigated the impact of different phytosanitary agents on the growth of P. lilacinum PL1, revealing both positive and negative effects depending on the agent application. Specifically, the pesticide AM, a secondary metabolite of Streptomyces avermitilis that is biodegradable by microorganisms (Abd-Elgawad 2020), had a negative effect on P. lilacinum PL1 growth. This suggests that P. lilacinum PL1 may not be able to degrade AM. Similarly, a previous study reported that the growth and sporulation of Trichoderma spp. were also suppressed by AM (Mayo-Prieto et al. 2022). Furthermore, other insecticides such as CM, TM, and DF, which act on the neurotransmitter systems of insects (Wakita 2011), did not inhibit the growth of P. lilacinum PL1. CuNPs, which are used as fertilizers and antibacterial (Rojas et al. 2021), did not affect the growth of P. lilacinum PL1, as observed in previous studies indicated that T. harzianum had increased sporulation in the supplement of CuNPs (Banik et al. 2017). However, the present study found that broad-spectrum triazole fungicides such as HC, CZ, and PP completely inhibited the expansion of P. lilacinum PL1 colonies (Zhou et al. 2022). Thus, using P. lilacinum PL1 in conjunction with pesticides may be an effective pest management technique, but the use of fungicides in tandem with P. lilacinum PL1 should be strictly regulated to prevent unintended consequences.
The efficient usage of P. lilacinum PL1 to fight against A. devastans nymph was further examined in laboratory and field conditions. The efficacy of P. lilacinum PL1 was evaluated by assessing the population of A. devastans nymphs subsequent to a 7-day treatment at a density of 1 × 107 conidia ml−1. These findings indicate that the utilization of P. lilacinum PL1 led to a noteworthy decrease in the population of A. devastans nymph, exhibiting an efficacy rate of 88.66% in vitro. The effectiveness demonstrated in this study is similar to the previous investigation, in which P. lilacinum PL1 was found to be successful in reducing B. tabaci nymph populations by a rate of 88.24% in vitro (Nguyen et al. 2023). Additionally, the results align with a previous study that demonstrated the efficacy of P. lilacinum XI-1 caused a lowering of 86.81% in adult whitefly populations within 7-day treatment (Sun et al. 2021). These findings demonstrated that P. lilacinum PL1 illustrates potential as a viable tool for pest management.
The efficacy of P. lilacinum PL1 in lowering the number of A. devastans nymphs in an okra plantation was also assessed in this study. The application of 1 × 107 conidia ml-1 of P. lilacinum PL1 resulted in a 64.46% reduction in nymph population after the first spraying, which further increased to 72.87% after the second spraying. These findings demonstrate the ability of P. lilacinum PL1 to effectively decrease A. devastans nymphs for a duration of up to 7 days. In the laboratory condition, AM was found to be more lethal to A. devastans nymphs than P. lilacinum PL1. However, under field conditions, the efficacy of AM dropped to 34.67%, significantly lower than the 72.87% mortality effect observed with P. lilacinum PL1. Sunlight, rising temperatures, and high soil moisture are all factors that might degrade AM and reduce its effectiveness under field conditions (Dionisio and Rath 2016). In the previous study, we indicated that P. lilacinum PL1 isolates thrive in tropical monsoon climates, as they can mature and sporulate at high temperatures up to 40 °C (Nguyen et al. 2023). Citrus psyllid (Diaphorina citri) populations may be lowered in the field by administering 1 × 107 conidia ml−1 of Isaria fumosorosea (Hoy et al. 2010). In addition, the A. biguttula population was reduced by 71.77–74.85%, when administrated with various biopesticides such M. anisopliae, B. bassiana, and V. lecanii (Janghel 2015). The results indicated that biopesticides are a viable alternative to chemical pesticides in managing sucking insects in okra. Integrated pest management involves combining chemical and biopesticide treatments for effective pest control. This study emphasizes the importance of exploring alternative methods for pest control to mitigate the negative impact of pesticides. Future studies are required to investigate the long-term effects of these interventions on the surrounding flora, soil, and ecological systems.
Utilization of microbial biopesticides, like P. lilacinum PL1, in sustainable agriculture is increasingly favored due to apprehensions regarding agrochemicals and the desire for organic food. The usage of cost-effective substrates for the production of P. lilacinum PL1 was crucial due to its contribution to sustainable pest management practices and economic benefits in developing countries. The present study indicated that rice and maize substrates were appropriated for enhancing the production of P. lilacinum PL1 conidia, particularly in an agricultural-oriented country like Vietnam. Furthermore, the effectiveness of P. lilacinum PL1 in reducing A. devastans nymph populations has been demonstrated in this study, including laboratory and field conditions. The promising findings highlight the potential of P. lilacinum PL1 as an effective and eco-friendly pest control agent, contributing to agriculture sustainable development. Nevertheless, the co-application of phytosanitary products might significantly affect the growth of P. lilacinum PL1, yielding either positive or negative efficacy in pest management. Therefore, it is advisable to exercise caution and regulate the application of fungicides in conjunction with P. lilacinum PL1 to avoid any adverse outcomes.
Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
Potato dextrose agar
Randomized complete blocks design
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The authors are especially grateful to Nguyen Tat Thanh University and HUTECH University for providing all the resources needed for this study.
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Nguyen Thi, H., Nguyen, Q.N., Dang Thi, N.Q. et al. Mass production of entomopathogenic fungi Purpureocillium lilacinum PL1 as a biopesticide for the management of Amrasca devastans (Hemiptera: Cicadellidae) in okra plantation. Egypt J Biol Pest Control 33, 85 (2023). https://doi.org/10.1186/s41938-023-00730-y
- Amrasca devastans
- Purpureocillium lilacinum
- Pest management